1. Field of Invention
The inventive subject matter relates to a recombinant nucleotide sequence and recombinant protein of Bartonella bacilliformis useful for the diagnosis of Oroya fever/verruga peruana due to Bartonella bacilliformis infection and for the immunization against Bartonella bacilliformis infection.
2. Background Art
Bartonella bacilliformis, is a small (0.2−0.5×10 μm), motile, pleomorphic coccobacillus. B. bacilliformis is the etiological agent of Oroya fever for the acute phase and verruga peruana for the chronic phase and is endemic in high altitude regions of the Colombian, Ecuadorian and Peruvian Andes. The acute phase is characterized by hemolytic anemia, fever, pallor and transient immuno-suppression (1). The second chronic phase is characterized by cutaneous vascular lesions.
Without antibiotics, mortality rates of 40–88% have been reported (2, 3). However, most of these fatalities are due to secondary infections by Salmonella or Toxoplasma. Appropriate antibiotic treatment reduces the fatality rate to about 9% (4, 5). Chloramphenicol is an effective treatment for both acute phase bartonellosis and secondary pathogens (4). This antibiotic, however, does not prevent the development of the eruptive phase of the disease. The chronic phase of the disease is best treated with streptomycin, rifampin or ciprofloxacin (4). Regardless of treatment, early diagnosis of the disease is extremely important for effective therapy. Furthermore, despite endemicity in South America, the threat of wide-spread bartonellosis is becoming an increasingly important health issue. Therefore, the disease is also becoming and increasing concern to overseas travelers.
A number of techniques are currently available for the diagnosis bartonella exposure and infection. A summary of these assays are listed in Table 1. Despite the array of available diagnostic methods, they all suffer from either being extremely laborious, expensive or time-consuming. These limitations are especially acute when the required test is conducted by facilities with limited resources or poor laboratory or clinical infrastructure.
Laboratory culture of B. bacilliformis is hampered by the bacterium's fastidious nature. Streaking whole blood onto heart-infusion agar plates supplemented with 5% rabbit blood is the best method for isolating the bacteria. Optimum growth is obtained by incubating each specimen at 28° C. without CO2 (4, 6). While highly sensitive, bacterial isolation requires two to five weeks to culture and identify the isolate. However, the prolonged incubation period predisposes the system to contamination and diminishes its
TABLE 1Current Diagnostic Techniques for BartonellosisPositivePredictiveMethodSensitivitySpecificityValueCommentIsolationRequires 2–5 weeksincubation, special media andtechniques (4, 6)Peripheral blood smearLow sensitivity in the chronicphase; Organisms can bedifficult to detect (4, 6, 7, 8)Giemsa thin smear36% 96%44%Low sensitivity; Organismscan be difficult to detect (6)ImmunostainAble to differentiate verrugaperuana from bacilliaryangiomatosis (8)PCR amplification of(9)231 bp genomic DNAsequencePCR amplification ofAble to detect 4 Bartonella16S rRNA gene codingspecies (10)regionPCR amplification ofAble to distinguish between B. quintana,the cell division (ftsZ)B. henselae, and B. bacilliformisgene(11, 12)PCR amplification ofAble to detect 6 Bartonellathe 16S–23S rRNAspecies (13)intergenic regionPCR assay targetingAble to detect 6 Bartonellariboflavin synthasespecies (14)gene (ribC)ELISACurrent technique requiresexpensive technology toprepare antigen (4, 15)Immunoblot with 65 kDa(16)antigenImmunoblot with30% for acute100%Not sensitive enough forGlycine extractedphase, 94%clinical use; Cross reactionsantigenchronic phasewith other Bartonella species(17)Immunoblot with70% acute phase,100%Cross reactions with othersonicated antigen94% chronicBartonella species (8, 17)phaseWestern Blot with FtsZAble to distinguish between B. quintana,proteinsB. henselae, and B. bacilliformis(11)IFA with irradiated82% 92%89%Low sensitivity for thewhole-cell antigenchronic phase(18)clinical utility when rapid diagnosis is needed or if limited laboratory infrastructure is available.
Peripheral blood smears and histopathologic studies are the most frequently used methods of diagnosis. In the acute phase, peripheral blood smears reveal the intra-erythrocytic bacilli of B. bacilliformis. Romanovski, Giemsa, and Wright stains can be used to visualize the bacteria (4, 7). Because the microorganisms are more difficult to visualize in the eruptive phase, final diagnosis is based on histopathological changes and is confirmed by Warthin-Starry staining (7). These staining methods are not desirable because the organisms are often not clearly identifiable, leading to potential misreading and therefore errors in diagnosis. Discrepancies between the interpretation of peripheral blood smears in the regional laboratory and a reference laboratory have been reported (8).
In 1999, Ellis and coworkers determined that Giemsa-stained peripheral smears are not as sensitive as antibody-based methods for the diagnosis of Bartonella (6). In this study, peripheral blood smears were stained with Giemsa and examined by light microscopy for the presence of the intra- or extra-erythrocytic coccobaccillary organisms. The sensitivity of this peripheral thin smear procedure was determined to be 36% when compared to bacterial isolation (6). Kosek and coworkers developed an immuno-stain to confirm the presence of B. bacilliformis in biopsied skin lesions (8). The biopsy samples were stained by the Steiner and Steiner silver method and then examined immunohistochemically. This immuno-stain method successfully labeled the bacteria in 100% of the lesions previously identified as verruga peruana on the basis of histopathologic appearance. This B. bacilliformis-specific immuno-stain can distinguish the miliary form of the verruga peruana from bacillary angiomatosis, a pathologically similar disease (8).
Bartonella can be detected and identified directly from clinical samples using PCR-derived assays. In 1992, Maass et al used PCR amplification of a 231 bp genomic Bartonella DNA sequence to detect B. bacilliformis in blood samples and skin biopsies (8). Matar and coworkers used a PCR method targeting the 16S rRNA gene coding region to detect four species of Bartonella (10). Kelly and coworkers designed a PCR-based diagnostic assay based on the cell division protein gene (ftsZ) that can successfully distinguish the genomes of B. henselae, B. quintana, and B. bacilliformis (11). This method requires only one round of PCR while avoiding the problems of specificity and consistency of previous studies. A later study confirmed that the ftsZ gene is a useful tool for detection and identification of Bartonella species (12). Jensen and coworkers developed a single-step PCR-based assay targeting the 16S–23S rRNA intergenic region that can detect six species of Bartonella, including B. bacilliformis (13). Johnson et al developed a genus-specific PCR assay using a single primer pair targeting the riboflavin synthase gene (ribC) able to detect six Bartonella species (14). While these molecular techniques offer high sensitivity and specificity, PCR assays require equipment and expertise not widely available in endemic areas.
Serologic assays are also used in the diagnosis of Bartonella infections. In 1988, Knobloch developed a B. bacilliformis-specific ELISA using liquid chromatography and photodiode array detection for the purification of the antigen (15). This technique requires expensive technology not widely available in endemic areas. In a subsequent study, the ELISA used to test for B. bacilliformis was positive in 89% of the culture positive patients (4). ELISAs are useful for large numbers of samples, are relatively inexpensive, and less subjective than other available methods. Additionally, ELISA, with the adequate antigen and assay design, are typically extremely sensitive.
Amano and coworkers used a recombinant B. bacilliformis 65 kDa protein in a Western blot in 1997 (16). Mallqui and coworkers developed two immuno-blot preparation methods for the diagnosis of bartonellosis (17). The antigen was prepared by either sonication of the whole cells or glycine extraction. On the basis of high sensitivity and specificity, two diagnostic bands were selected for each preparation method, at 17-kDa and 18-kDa for the sonicated antigen and at 16-kDa and 18-kDa for the glycine extracted antigen. Antibodies to several different bacteria, including C. psittaci and Brucella, cross-reacted with these B. bacilliformis antigens. The high rate of cross-reactivity (40%) with Brucella is important because of the similarity between the symptoms of acute phase bartonellosis and Brucellosis. The glycine extraction method is not suitable for use in diagnosis due to its higher rate of cross-reaction and insufficient detection of the acute phase. The immunoblot using the sonicated antigen was 70% sensitive for acute disease, 94% sensitive in identifying chronic bartonellosis and 100% specific. Because it is both highly sensitive and specific, the sonicated immunoblot preparation method is suitable for use in endemic regions. This sonicated-immunoblot preparation method was used in a serological survey after an outbreak of bartonellosis in Peru (8). In 2001, Maguina and coworkers performed ELISA and Western immunoblots (4). The Western immunoblot was positive in 100% of culture-positive patients. All of the patients (n=12) with eruptive phase Bartonellosis had positive ELISA and Western immunoblots.
Kelly and coworkers developed a serological test to distinguish among B. quintana, B. henselae, and B. bacilliformis based on differences in the FtsZ gene (11). Kelly et al synthesized peptides corresponding to the regions of greatest divergence in this gene and injected them into rabbits to generate species-specific antisera. In an immunoblot, the rabbit antisera reacted only with the FtsZ protein from the specific Bartonella species. When the extracts were incubated with antisera to the heterologous peptide sequence, no immunoreactive protein of the size of FtsZ was detected. These results suggest that the differences in these sequences could be used as species-specific antigens for serological diagnosis.
The first successful IFA for the detection of antibodies to B. bacilliformis was developed in 2000(17). The IFA tests uses irradiated whole-cell antigen preparation co-cultivated with Vero cells (18). This IFA test was 82% sensitive in detecting the antibodies in acute-phase blood samples in laboratory-confirmed bartonellosis patients. The IFA was positive for 93% of the convalescent-phase cases (18). The specificity of this IFA is 92%, high enough for epidemiological use. This genus-specific IFA does not react with patient serum with other well-described diseases such as brucellosis, typhoid fever, lyme disease, dengue fever, ehrichiosis, and secondary syphilis. Indirect fluorescence assays are time consuming, costly and require expertise to achieve reproducible results. Serodiagnosis by indirect fluorescence assay is not desirable because of inter-observer variability and the potential for misdiagnosis due to antigen cross-reactivity.
Because B. bacilliformis is the causative agent of Oryoya fever and veruga peruana, where mortality can be exceptionally high, improved methods for B. bacilliformis exposure and infection are needed over existing procedures. Improvements in speed of assay and sensitivity are likely to significantly enhance early diagnosis and therefore administration of treatment with life-saving results. Additionally, a reduction in the required reagents and facilities will expand the ability to conduct the diagnostic methods to facilities with more limited resources, obviating the need to send samples long distances to more complete laboratories for testing thus further improving the speed of diagnostic results.